Association of leptin receptor polymorphisms with susceptibility of non‐small cell lung cancer: Evidence from 2249 subjects

Abstract Non‐small cell lung cancer (NSCLC) is increasing dramatically. It is believed that energy metabolism‐related genes could play an important role in etiology of NSCLC. In this study, we sought to assess the correlation between three LEPR single nucleotide polymorphisms (rs1137101, rs1137100 and rs6588147) with NSCLS susceptibility. In total, 1193 NSCLC cases and 1056 controls were included. SNPscan™ genotyping method was used to analyze the genotypes of LEPR polymorphisms. Compared to rs6588147 GG in LEPR gene, this study identified a protective role of LEPR rs6588147 GA and GA/AA for the occurrence of NSCLC (GA vs. GG [p = 0.021] and GA/AA vs. GG [p = 0.030]). As well, we found that a protective role of LEPR rs6588147 for the occurrence of non‐SCC subgroup (p < 0.05). By logistic regression analysis, we found that the rs6588147 A allele related genotypes might play a protective role for the occurrence of NSCLC in drinking, BMI ≥24 kg/m2, smoking and male subgroups. We also found that the rs1137101 A allele related genotypes played a protective role for the occurrence of NSCLC in male, younger participants (under 59 years) and overweight/obesity (BMI ≥24 kg/m2) subgroups. We found that LEPR Ars1037100Ars1037101Ars6588147 haplotype might play a protective role for the occurrence of NSCLC (p = 0.013). In addition, our findings indicated that LEPR rs1137100 G>A SNP might increase the risk of lymph node metastases (p = 0.038). This study highlights that LEPR rs6588147, rs1137101 genotypes and LEPR Ars1037100Ars1037101Ars6588147 haplotype are correlated with the occurrence of NSCLC. LEPR rs1137100 G>A SNP increases the risk of lymph node metastases.

ogy of NSCLC.In this study, we sought to assess the correlation between three LEPR single nucleotide polymorphisms (rs1137101, rs1137100 and rs6588147) with NSCLS susceptibility.In total, 1193 NSCLC cases and 1056 controls were included.SNPscan™ genotyping method was used to analyze the genotypes of LEPR polymorphisms.Compared to rs6588147 GG in LEPR gene, this study identified a protective role of LEPR rs6588147 GA and GA/AA for the occurrence of NSCLC (GA vs. GG [p = 0.021] and GA/AA vs. GG [p = 0.030]).As well, we found that a protective role of LEPR rs6588147 for the occurrence of non-SCC subgroup (p < 0.05).By logistic regression analysis, we found that the rs6588147 A allele related genotypes might play a protective role for the occurrence of NSCLC in drinking, BMI ≥24 kg/m 2 , smoking and male subgroups.We also found that the rs1137101 A allele related genotypes played a protective role for the occurrence of NSCLC in male, younger participants (under 59 years) and overweight/obesity (BMI ≥24 kg/m 2 ) subgroups.We found that LEPR A rs1037100 A rs1037101 A rs6588147 haplotype might play a protective role for the occurrence of NSCLC (p = 0.013).
In addition, our findings indicated that LEPR rs1137100 G>A SNP might increase the risk of lymph node metastases (p = 0.038).This study highlights that LEPR 1

| INTRODUCTION
With a promoting lung cancer (LC) incidence worldwide, 1 LC is the leading cause of cancer-related death.Non-small cell lung cancer (NSCLC), the most common subtype of lung cancer (LC), is also increasing dramatically in China. 2 The increasing occurrence of LC might be correlated with some environmental factors (e.g., exposure to tobacco, indoor radon level, air pollution, etc.).Additionally, inactive lifestyle, central obesity and type 2 diabetes mellitus (T2DM) may be implicated in the development of LC. [3][4][5][6][7][8][9] Over the past half-century in China, significant lifestyle and daily diet have occurred, leading to a continuous increase in the rates of overweight and obesity. 102][13][14] It is believed that metabolism-related genes play an important role in the etiology of LC.
Leptin (LEP) is a hormone, which is produced by the OB gene.It may be involved in regulating food intake and energy spending.A higher LEP level has been found in overweight and obesity individuals. 15LEP receptor (LEPR), a transmembrane cytokine receptor, interacts with LEP and influences the food intake and energy metabolism.Some previous investigations reported that LEPR expression was associated with the proliferation, immune, inflammatory and neoangiogenesis. 16,17It was believed that dietary factors were answerable for about a third of overall malignancies. 18 number of publications suggested that diet and obesity might contribute to the susceptibility of LC. [19][20][21][22] Li et al. reported that the LEP and LEPR were more highly expressed in NSCLC cells than in normal lung tissue, which could promote NSCLC by activating the MAPK/ERK and PI3K/AKT pathways.23 The level of LEPR could be a useful biomarker for NSCLC prognosis.Xu et al. also suggested that LEPR expression increased in the tissues of NSCLC than in normal lung tissue.24 Thus, the LEPR could play a vital role in the etiology and development of NSCLC.
Single nucleotide polymorphism (SNP) is the most common type of variations.Several functional SNPs of LEPR have so far been explored for their roles in contributing to the occurrence of cancer.The rs1137100 and rs1137101 SNPs in LEPR gene, G → A mutation, were two common missense SNPs.It is well known that missense SNP may lead to an amino acid change in LEPR.Li et al. first studied the association of rs1137100 and rs1137101 SNPs in LEPR with NSCLC susceptibility.Compared with AA, they found that LEPR rs1137101 GG, increased 3.12-fold risk of NSCLC in Chinese populations. 25And they also observed that LEPR rs1137101 GG carriers of NSCLC patients might have a poor survival than rs1137101 AG and AA carriers. 25ubsequently, in Caucasians, a case-control study explored the relationship of rs1137101 with LC risk and reported null association. 26Rs6588147 G>A in LEPR, an intron variant, was found to be correlated with a susceptibility of colon carcinoma in Caucasians. 27Recently, in Asian populations, the relationship of rs6588147 in LEPR with cancer risk was reported with inconsistent findings.Qiu et al. found that LEPR rs6588147 was associated with the increased risk of esophageal squamous cell carcinoma. 28However, Lin et al. and Zhang et al. reported that LEPR rs6588147 decreased the risk of colorectal cancer and hepatocellular carcinoma, respectively. 29,30Thus, the correlation of LEPR rs6588147 with the risk of cancer was conflicting.Here, the rs1137101, rs1137100 and rs6588147 loci in LEPR gene were selected based on the previous studies mentioned above.We performed an investigation to assess the correlation of LEPR SNPs with NSCLC susceptibility.

| Statement of ethics
Ethics Committee of Fujian Medical University Union Hospital approved this investigation.Each participation was informed the aim of this study.The research has been carried out in accordance with the World Medical Association Declaration of Helsinki, and that all subjects provided informed consent.

| Study populations
The NSCLC patients were recruited between January 2014 and June 2018 at Fujian Medical University Union Hospital (Fujian Province, China) and Nanjing Medical University Zhenjiang Medical College (Jiangsu Province, rs6588147, rs1137101 genotypes and LEPR A rs1037100 A rs1037101 A rs6588147 haplotype are correlated with the occurrence of NSCLC.LEPR rs1137100 G>A SNP increases the risk of lymph node metastases.

K E Y W O R D S
LEPR, non-small cell lung cancer, obesity, overweight, polymorphism | 3 of 12 TANG et al.
China).All NSCLC cases were residents of Fujian, Jiangsu Provinces and the surrounding areas.When enrollment was carried out, the following criteria were used: (1) without history of other malignancy, (2) before any treatment for NSCLC, and (3) Chinese Han ethnicity.The following criteria were used for exclusion: (1) with a second primary malignancy, (2) after radiotherapy and chemotherapy treatment, and (3) diagnosed as small cell lung cancer cases.NSCLC stage was classified according to AJCC 8.0 version (2017) of LC.Of the 1193 NSCLC patients, 182 (15.26%) were squamous cell carcinoma (SCC), and 1011 (84.74%) were non-SCC.In addition, at the same time, 1056 healthy controls were included from the institutions mentioned above.Controls were full-matched with NSCLC patients on age and sex (p = 0.330 and 0.425, respectively).Healthy control subjects were Chinese Han populations who were unrelated to NSCLC patients.In this study, body mass index (BMI) was assessed by height and weight, which were collected by two authors.BMI ≥24 kg/m 2 was considered as the criteria of overweight and obesity. 31,32Subjects who consumed one cigarette per day for more than 1 year were considered as "smokers". 33ndividuals who drank more than three times a week for longer than 6 months were considered to be "alcohol users". 33Each participant completed a questionnaire and donated 2 mL blood sample.The information including smoking, drinking, age, sex, ethnicity, height, weight and results of pathology was collected.

| Genotyping
The blood sample was stored with EDTA anticoagulant.A "Salting-out" method was used to extract DNA by using the DNA Purification Kit (Promega, Madison, USA).A 260/280 ratio checking was used to confirm the purity of each DNA sample.The rs1137101, rs1137100 and rs6588147 genotypes were determined by SNPscan™ method. 34,35In ABI 2720 thermal cycler, the ligation reaction was conducted.Then a 48-plex fluorescence PCR was carried out.The PCR products were analyzed by capillary electrophoresis in an ABI 3730XL sequencer.The information of LEPR genotypes was read by GeneMapper 4.1 software (Applied Biosystems, Foster City, CA, USA).For each of LEPR SNPs, 112 DNA samples were chosen randomly and assayed the LEPR genotypes to confirm the veracity of our results.And consistent LEPR genotypes were found.

| Statistical analysis
Student's t-test was harnessed to treat continuous variable comparison.Intergroup difference of LEPR genotypes, BMI, alcohol consumption, sex, smoking and age was determined by chi-square test.By logistic regression analysis, crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the relationships in homozygous, dominant, heterozygous and recessive models in overall or subgroup analysis.All statistical analyses were performed by using SAS 9.4 software (SAS Institute, Inc.).7][38][39][40][41] An internet haplotype construction method, SHESIS software (http:// analy sis.bio-x.cn/ myAna lysis.3][44][45] A p < 0.05 was used as the criterion of the significance level.Additionally, Bonferroni correction was applied to adjust for multiple testing. 46 3 |RESULTS

| Characteristics
Table 1 shows the information and baseline characteristic of 1193 NSCLC cases and 1056 controls.Age and sex were well matched (p = 0.330 and p = 0.425).Of the NSCLC patients, 182 were squamous cell carcinoma (SCC), and 1011 were non-SCC.The stage of NSCLC patients was classified according to AJCC 8.0 version (2017).Among them, 703 were stage I, 87 were stage II, 222 were stage III and 181 were stage IV.We summarized the chromosomal location, region, and minor allele frequency (MAF) information in Table 2.For LEPR SNPs, success rate of genotyping was more than 99%.The value of HWE was also calculated and shown in Table 2 (all p > 0.05).MAFs of LEPR rs1137101, rs1137100 and rs6588147 (0.124, 0.159 and 0.151) could be obtained in Table 2, which were analogous to the data of the Chinese populations.Raw data of genotypes and characteristics were summarized in Table S1

| Association of LEPR polymorphisms with NSCLC
The LEPR rs1137101, rs1137100 and rs6588147 genotypes in overall NSCLC cases and different pathological subtype are shown in Table 3.The LEPR genotypes in control group are also summarized in Table 3.
In overall comparison, we did not find any relationship of LEPR rs1137101 and rs1137100 loci with the risk of NSCLC.
As summarized in Table 7, we found that the rs1137100 A allele did not influence the occurrence of NSCLC in different subgroup.

| Association of LEPR polymorphisms with lymph node metastases and stage
We analyzed the association of LEPR polymorphisms with lymph node metastases and stage.We found that LEPR rs1137100 G>A SNP might increase the risk of lymph node metastases of NSCLC (OR = 1.35, 95% CI = 1.02-1.79,p = 0.038, Table 9).

| DISCUSSION
LEPR is a transmembrane cytokine receptor, which combines to LEP and regulates the food intake and energy metabolism.8][49] In this study, we conducted a case-control study and explored the role of LEPR SNPs in the development of NSCLC.Rs6588147, an intron SNP, located on Chromosome 1: 65469811 (GRCh38). 27The function of LEPR rs6588147 was unknown.Our investigation showed that the A allele of rs6588147 could decrease the susceptibility of NSCLC in overall comparison (GA vs. GG: adjusted p = 0.021 and AA/GA vs. GG: adjusted p = 0.030).Additionally, the role of decreasing the susceptibility of NSCLC was also confirmed among drinking, smoking and male subgroups, even a Bonferroni correction applied to adjust for multiple testing.Lin et al. found that this SNP decreased colorectal cancer risk in the male subgroup, 29 which was similar to our findings.Slettery and colleagues first explored the relationship of LEPR rs6588147 with the occurrence of colon cancer and indicated that A allele of rs6588147 had a tendency to reduce the occurrence of colon cancer in Caucasians. 27Additionally, in Chinese populations, previous studies reported that rs6588147 locus in LEPR gene was associate with a lower susceptibility of colorectal cancer and hepatocellular carcinoma. 29,30Our observations were similar to them.However, several investigations suggested that A allele of rs6588147 had a higher susceptibility of esophageal squamous cell carcinoma and breast cancer. 28,50Due to the limited sample sizes, the role of rs6588147 on cancer susceptibility may be controversial.In the future, more investigations focusing on environmental factors are needed.LEPR Arg223Gln (rs1137101), a G → A variation on exon 6, is a missense SNP. 51The role of LEPR rs1137101 in the development of cancer has been studied. 52,53In the extracellular region, this missense polymorphism results in an Arg→Gln substitution. 51oothby-Shoemaker et al. reported that the lower FGFR1 and LEPR expression could delay cancer progression via JAK2 signaling. 54Stefan et al. suggested that A allele of LEPR rs1137101 might reduce the binding of LEP with LEPR. 55A recent investigation also identified that the AA genotype of LEPR rs1137101 with a lower binding ability to its ligand could decrease the activity of Ras/ERK1/2 and JAK2/ STAT3 the signaling pathways and played a protective role in carcinogenesis. 56Two case-control studies had been performed to investigate the relationship of LEPR Arg223Gln with the susceptibility of NSCLC in different ethnicities.Li et al. reported that A allele (223Gln) of LEPR rs1137101 reduced the susceptibility of NSCLC in Chinese population. 25In addition, in Caucasians, Unsal et al. found that combined genotypes of LEP 2548G>A and LEPR rs1137101 SNPs might have a protective effect against the occurrence of LC. 26 In the present investigation, the relationship between rs1137101 and a decreased susceptibility of NSCLC was found in male, younger (<59 years) and overweight/obesity (BMI ≥24 kg/m 2 ) subgroups, which was similar to previous studies.The association of LEPR rs1137100 SNP with risk of cancer were inconsistent.In this study, we found that there was null association between LEPR rs1137100 SNP with risk of NSCLC.However, we found that LEPR rs1137100 G>A SNP might increase the risk of lymph node metastases (p = 0.038).To the best of our knowledge, this study was the first investigation in which the relationship of LEPR rs1137100 G>A SNP with lymph node metastases was found.In the future, more studies should be conducted to explore the potential association of LEPR rs1137100 SNP with lymph node metastases.
In the future, more studies should be conducted to explore the potential association between LEPR rs1137100 and the risk of NSCLC.
As listed in Table 7, compared to LEPR G rs1037100 G rs1037101 G rs6588147 haplotype, we found that the frequency of LEPR A rs1037100 A rs1037101 A rs6588147 haplotype was significantly low in NSCLC group (p = 0.013), which is similar to the previous findings. 57To the best of knowledge, we first focused on the correlation of LEPR haplotypes with NSCLC susceptibility.These findings suggested that LEPR A rs1037100 A rs1037101 A rs6588147 haplotype could be used in screening high-risk populations for NSCLC.There were some limitations in the present study.Firstly, for lack of data on lifestyle and dietary factors (low intake of fruit and veggies, poor diet, physical inactivity, high outdoor ambient air pollution, and cooking oil fume, etc.), we could not explore an interaction of LEPR rs1137101, rs1137100 and rs6588147 polymorphisms with these conditions.Secondly, although the sample sizes were relatively large, the number of the participants in some subgroups was limited.Thirdly, given the hospital-based design, the selection bias might have happened.Fourthly, only three functional SNPs of LEPR gene were studied in this investigation.Other vital LEPR SNPs should not been ignored.Fifthly, diabetes mellitus and family history of participants were not collected.In future studies, these vital risk factors should not be overlooked.Sixthly, when designed this case-control study, we did not consider the association between LEPR SNPs and EGFR status of NSCLC.Finally, this investigation was carried out in China.In the future, more well-designed studies should be performed to explore the association of LEPR SNPs with the risk of NSCLC in other ethnicities.
In conclusion, the present study highlights that LEPR rs6588147 and rs1137101 genotypes and LEPR Ars1037100Ars1037101Ars6588147 haplotype are correlated with the occurrence of NSCLC.LEPR rs1137100 G>A SNP increases the risk of lymph node metastases.Additionally, rs6588147 A allele could decrease the susceptibility of NSCLC was also confirmed among drinking, smoking and male subgroups, even a Bonferroni correction applied to adjust for multiple testing.The current investigation attempted to point out the potential contribution of LEPR SNPs to NSCLC etiology and also for screening high susceptibility individuals for prevention and early detection of NSCLC.To further explore LEPR SNPs and NSCLC risk, more case-control studies in diverse ethnicities along with functional and expressional characterizations of the LEPR SNPs are needed.
T A B L E 1Note: Bold values are statistically significant (p < 0.05).Abbreviations: BMI, body mass index; NSCLC, non-small-cell lung cancer; SCC, squamous cell carcinoma.aTwo-sidedχ 2 test and student's t-test.T A B L E 2 a http://www.regulomedb.org/.Abbreviations: HWE, Hardy-Weinberg equilibrium; MAF, minor allele frequency.T A B L E 3The frequencies of LEPR rs1137100, rs1137101 and rs6588147 polymorphisms in NSCLC patients and controls.TA B L E 4Abbreviations: NSCLC, non-small-cell lung cancer; SCC, squamous cell carcinoma.a Adjusted for age, sex, smoking, drinking and body mass index.
Stratified analyses between LEPR rs1137101 G>A polymorphism and NSCLC risk by sex, age, BMI, smoking status and alcohol consumption.
T A B L E 5 aFor LEPR rs1137101 G>A, the genotyping was successful in 1184 (99.25%)NSCLC cases, and 1052 (99.62%) controls.b Adjusted for multiple comparisons [age, sex, smoking status, BMI and alcohol consumption (besides stratified factors accordingly)] in a logistic regression model.
Stratified analyses between LEPR rs6588147 G>A polymorphism and NSCLC risk by sex, age, BMI, smoking status and alcohol consumption.
T A B L E 6 Stratified analyses between LEPR rs1137100 G>A polymorphism and NSCLC risk by sex, age, BMI, smoking status and alcohol consumption.
With the order of LEPR rs1137100 G>A, rs1137101 G>A and rs6588147 G>A in gene position.Compared with LEPR G rs1037100 G rs1037101 G rs6588147 haplotype, LEPR A rs1037100 A rs1037101 A rs6588147 haplotype might decreased the risk of NSCLC.Analyses of LEPR rs1137100, rs1137101 and rs6588147 polymorphisms with lymph node metastases and stage.Bold values are statistically significant (p < 0.05).
Note:Note: a Adjusted for age, sex, smoking, drinking and body mass index.